African Journal of Pharmacy and Pharmacology Vol. 6(40), pp. 2802-2807, 29 October, 2012 Available online at http://www.academicjournals.org/AJPP DOI: 10.5897/AJPP12.295 ISSN 1996-0816 ©2012 Academic Journals

Full Length Research Paper

Chemical assessment of wild Paris rhizome from Southwest China

Jinyu Zhang1, Tao Shen2, Yuanzhong Wang1, Ji Zhang1, Yundong Shi2 and Hang Jin1*

1Institute of Medicinal Plants, Yunnan Academy of Agricultural Sciences, Kunming 650223, China. 2College of Resource and Environment, Yuxi Normal University, Yuxi 653100, China.

Accepted 8 May, 2012

Chonglou is a well known traditional Chinese medicine for anti-cancer and traumatic injuries. The major sources of Chonglou are from genus Paris. The present investigation aimed to determine contents of active steroidal saponins in Paris plants collected from different locations of Southwest China. The results showed that polyphyllin I and polyphyllin VII could be detected in almost all samples. However, polyphyllin II and polyphyllin VI were not detected in the research. The results indicated that besides Paris polyphylla var. yunnanensis and P. polyphylla var. chinensis, used as medicine traditionally, other Paris species in Southwest China also have medicinal value. Ranges of bioactive compounds in P. polyphylla var. yunnanensis from different parts of the plants were wide. Coefficient of variation of polyphyllin I, polyphyllin II, polyphyllin VII, and total steroidal saponins were 53.4132, 43.9341, 88.2673, and 60.9082%, respectively. Varieties of compounds type and contents in Paris plants would be related to habitats.

Key words: Paris genus, polyphyllin, variation, high performance liquid chromatography (HPLC), Southwest China.

INTRODUCTION

Traditional Chinese medicine (TCM) is getting more about 24 species distributed from Europe to Eastern Asia. understood and popular worldwide in different medical There are 22 Paris species in China. The diversity center purpose. Nowadays, TCM is playing an important role in of this genus is located in Southwest China (Li, 1986; Li, healthcare system in several Asian countries (Elena et 1998). The rhizomes of several species of Paris, such as al., 2010). Medicinal plants are an essential part of TCM. Paris polyphylla var. yunnanensis and P. polyphylla var. These plants have been used to treat various diseases chinensis are famous traditional Chinese herb named for more than 8000 years (Drǎsar and Moravcova, 2004). Chonglou. Paris plants as medicine in folk of China have Recently, a large number of bioactive compounds were a history of thousands of years (Li, 1998), and are widely found from medicinal plants and these compounds are used to treat parotitis, fractures, snakebite, hemostasis, the key compounds of TCM development and utilization and abscess (Committee for the Pharmacopoeia of P. R. (Calvin and Chen, 2011). Steroidal saponins were China). It is also the main raw material of some well-know isolated from various plants. These chemical compounds Chinese patent drug, such as “Yunnan Baiyao”, have bioactivities for anticancer, antibacterial, anti- „GongXue Ning‟, etc (Li, 1998; Zhang et al., 2011). hypertensive, antivirus, relieve inflammation, sedative, The quality of Chonglou is been measured by bioactive and anthelmintic activity. Hence, steroidal saponins are compound contents. Polyphyllin I, polyphyllin II, termed as important natural products (Deng et al., 1999; polyphyllin VI, and polyphyllin VII are a group of steroidal Sun et al., 2002). saponins, and important components of Paris genus Paris, belonging to the family Trilliaceae, is a genus of (Committee for the Pharmacopoeia of P.R. China, 2010; Zhang et al., 2011). Previous study revealed that polyphyllin has powerful pharmacological activities on homeostatic, spermicidal, and analgesic (Li, 1998). In *Corresponding author. E-mail: [email protected]. Tel: +86 recent years, researchers found that polyphyllin I was a 0871 8060004. potential anti-cancer drug as the functions of cytotoxic Zhang et al. 2803

Table 1. Calibrations of polyphyllin I, polyphyllin II, polyphyllin VI and polyphyllin VII.

Standard Regression equation R2 Polyphyllin I y=83,677,600.0000x-82,415.3333 0.9909 Polyphyllin II y=226,795,736.8421x-43,412.2631 0.9985 Polyphyllin VI y=212,038,400.0000x-26,967.53333 0.9971 Polyphyllin VII y=239,018,066.6666x-10,685.3111 0.9994

PolyphyllinⅠ alcohol under reflux in water bath for 45 min. 1.5 ml of the extract PolyphyllinⅥ was transferred into 2 ml centrifuge tube, then centrifuged at 16,000 rpm for 10 min after cooled to room temperature. PolyphyllinⅦ PolyphyllinⅡ

Chemicals and reagents

The standards (polyphyllin I, polyphyllin II, polyphyllin VI, and polyphyllin VII) were purchased from the National Institute for Control of Pharmaceutical and Biological Products (Beijing, China). Acetonitrile (high performance liquid chromatography (HPLC) grade) was purchased from TEDIA (Ohio, USA). Purified water (HPLC grade) was produced by Milli-Q system (Massachusetts, USA). Other reagents were all of analytical grade. Time (min)

Figure 1. Standard steroidal saponins: polyphyllin I (34.828 min), polyphyllin II (32.384 min), polyphyllin VI (23.048 min), and Chromatographic conditions polyphyllin VII (20.980 min). HPLC system (Shimadzu Technologies, Kyoto, Japan) was equipped with the Workstation software class-VP (Shimadzu and apoptosis (Lee et al., 2005; Ong et al., 2008; Siu et Technologies) and composed of HPLC-10 Integrator, HPLC- 10ATVP pump, and SPD-M10A VP detector (DAD). All al., 2008). chromatographic separations were carried out on a Shim-pack VP- P. polyphylla var. yunnanensis and P. polyphylla var. ODS C18 (150 × 4.6 mm, particle size: 5 μm) from Shimadzu chinensis are the main materials of medicine in the official (Kyoto, Japan). Mobile phase solvents A (water) and B (acetonitrile) records (Committee for the Pharmacopoeia of P.R. China, were applied. The flow-rate: from 70:30 (A:B) to 45:45 in 20 min, 2010). However, many other Paris plants could be found from 45:45 to 30:60 in 10 min, and from 30:60 to 70:30 in 10 min. in markets in China. To study contents and distribution of The flow-rate was 1.0 ml/min, injection volume: 10 μl, column temperature: 25°C. Detection wavelength: 210 nm. important bioactive compounds in Paris genus might help us to understand differences of Paris medicinal materials from different source and to improve conservation and Preparation of standard solutions utilization of Paris genus resources. The aim of this study was to describe the variation of the Pure standards of polyphyllin I, polyphyllin II, polyphyllin VI, and contents of main active compounds (polyphyllin I, polyphyllin VII were introduced as external standards in the polyphyllin II, polyphyllin VI, and polyphyllin VII) in some analysis. Standard solutions with different concentrations were medicinal Paris plants from Southwest China. dispensed in methanol. All standard solutions for HPLC were filtered using 0.25 μm syringe filter before injection.

MATERIALS AND METHODS RESULTS AND DISCUSSION Plant Qualitative analysis The plant materials included 12 species collected from 43 locations in wild habitats of Yunnan, Guizhou, and Guangxi in Southwest China (Table 1). Standard of polyphyllin I, polyphyllin II, polyphyllin VI, and polyphyllin VII were tested on HPLC. Six repeated measurements were then given to each sample. The Preparation of samples range of standard curve of polyphyllin I and polyphyllin II The rhizomes of Paris plants were dried under the temperature of was 0.01 to 1. 0 mg/ml. The range of standard curve of 50°C and ground to fine powder using a pestle mortar. polyphyllin VI and polyphyllin VII was 0.002 to 0.1 mg/ml. Chromatograms and calibrations are shown in Figure 1 and Table 2. Extraction Table 3 shows the HPLC analysis results. Polyphyllin I The rhizomes powder (0.5000 g) was extracted with 25 ml of 98.0% and VII have been detected in almost all samples however, 2804 Afr. J. Pharm. Pharmacol.

Table 2. Sample locations.

Sample number Species Location 1 Paris caobangensis Jinchang, Wenshan, Yunnan 2 P. cronquistii Yangchang, Bijie, Guizhou 3 P. cronquistii Malipo, Wenshan, Yunnan 4 P. cronquistii var. xichouensis Jinchang, Wenshan, Yunnan 5 Paris delavayi var. petiolata Jingxi, Baise,Guanxi 6 P. fargesii Yangchangzheng, Bijie, Guizhou 7 P. fargesii Guanteng, Bijie, Guizhou 8 P. fargesii Dayixiang, Bijie, Guizhou 9 P. mairei Maanshan, Dali, Yunnan 10 P. polyphylla Maanshan, DaLi, Yunnan 11 P. polyphylla Lameirong, Lijiang, Yunnan 12 P. polyphylla var. alba Machang, DaLi, Yunnan 13 P. polyphylla var. chinensis Maguan, Wenshan, Yunnan 14 P. polyphylla var. chinensis Jingxi,Baise,Guanxi 15 P. polyphylla var. chinensis Napo,Baise,Guanxi 16 P. polyphylla var. pseudothib Jiaozixueshan, Dongchuan, Yunnan 17 P. polyphylla var. yunnanensis Wadu, Baoshan, Yunnan 18 P. polyphylla var. yunnanensis Tuanjiexiang, Kunming, Yunnan 19 P. polyphylla var. yunnanensis Tuanjiexiang, Kunming, Yunnan 20 P. polyphylla var. yunnanensis Bagaxiang, Wenshan, Yunnan 21 P. polyphylla var. yunnanensis Wuding, Chuxiong, Yunnan 22 P. polyphylla var. yunnanensis Mojiang, Puer, Yunnan 23 P. polyphylla var. yunnanensis Pingbaxiang, Wenshan, Yunnan 24 P. polyphylla var. yunnanensis Gongshan, Nujiang, Yunnan 25 P. polyphylla var. yunnanensis Lancanglinchang, Puer, Yunnan 26 P. polyphylla var. yunnanensis Xinping, Yuxi, Yunnan 27 P. polyphylla var. yunnanensis Zhengxiong, Zhaotong, Yunnan 28 P. polyphylla var. yunnanensis Dazhai, Lincang, Yunnan 29 P. polyphylla var. yunnanensis Laowo, Nujiang, Yunnan 30 P. polyphylla var. yunnanensis Changlinjie, Wenshan, Yunnan 31 P. polyphylla var. yunnanensis Jingu, Puer, Yunnan 32 P. polyphylla var. yunnanensis Kafang, Gejiu, Yunnan 33 P. polyphylla var. yunnanensis Beicheng, Yuxi, Yunnan 34 P. polyphylla var. yunnanensis Liangwangshan, Kunming, Yunnan 35 P. polyphylla var. yunnanensis Lianhuatang, Wenshan, Yunnan 36 P. polyphylla var. yunnanensis Zhengyuan, Puer, Yunnan 37 P. polyphylla var. yunnanensis Baihualing, Nujiang, Yunnan 38 P. polyphylla var. yunnanensis Yanglin, Kunming, Yunnan 39 P. polyphylla var. yunnanensis Dimaluo, Nujiang, Yunnan 40 P. polyphylla var. yunnanensis Qishe, Xingyi, Guizhou 41 P. polyphylla var. yunnanensis Lanping, Nujiang, Yunnan 42 P. polyphylla var. yunnanensis Menghai, Xishuangbanna, Yunnan 43 P. polyphylla var. yunnanensis Yunxian, Lincang, Yunnan 44 P. polyphylla var. yunnanensis Gulinqing, Wenshan, Yunnan 45 P. polyphylla var. yunnanensis Zhuanlong, Kunming, Yunnan 46 P. vietnamensis Nansan, Lincang, Yunnan 47 P. cronquistii var. xichouensis Napo,Baise,Guanxi 48 P. mairei Fenshuiling, Nujiang, Yunnan 49 P. polyphylla var. yunnanensis Mengze, Xishuangbanna, Yunnan 50 P. polyphylla var. yunnanensis Ruili, Dehong, Yunnan 51 P. vietnamensis Mengze, Xishuangbanna, Yunnan Zhang et al. 2805

Table 3. Contents of detected compounds in Paris plants (mg/g).

Sample number Polyphyllin I Polyphyllin II Polyphyllin VI Polyphyllin VII Total steroidal saponins 1 8.61 - - 0.49 9.11 2 11.53 - - 0.14 11.67 3 - - - 0.15 0.15 4 6.74 1.52 - 0.14 8.40 5 3.32 - - 1.63 4.95 6 4.45 1.23 - - 5.67 7 12.57 0.90 - 0.15 13.63 8 - 2.66 - 0.43 3.09 9 10.03 1.34 - 0.43 11.81 10 15.76 2.55 - 0.16 18.47 11 8.41 0.91 - 1.28 10.60 12 9.67 1.69 - 0.83 12.19 13 27.04 - - 4.32 31.36 14 11.00 1.67 - 0.25 12.91 15 12.09 1.24 - 0.33 13.66 16 31.03 1.91 - 0.40 33.34 17 3.43 - 2.97 0.15 6.55 18 2.90 - - 0.43 3.33 19 9.24 1.49 - - 10.73 20 5.00 1.65 - 0.57 7.22 21 6.92 - - 0.68 7.60 22 12.41 1.59 - 0.15 14.15 23 13.04 4.69 - 0.69 18.42 24 16.37 2.27 - 0.17 18.80 25 2.91 - - 0.25 3.16 26 13.44 3.41 - 0.51 17.36 27 3.17 - - 2.07 5.24 28 19.80 2.04 - 0.24 22.07 29 - - - 0.16 0.16 30 - 3.75 - 0.42 4.18 31 18.35 1.90 - 0.42 20.66 32 14.52 1.47 - 0.25 16.24 33 3.81 - - 0.62 4.43 34 3.43 - - 1.54 4.97 35 - 2.59 - 0.30 2.89 36 11.63 3.24 - 0.87 15.74 37 4.99 1.74 - 0.17 6.90 38 11.04 2.93 - 0.74 14.71 39 9.85 - - 0.43 10.28 40 7.60 4.81 - 0.48 12.90 41 4.39 - - 0.54 4.93 42 15.60 4.37 - 0.25 20.22 43 10.57 3.85 - 0.15 14.56 44 15.02 - - 0.21 15.23 45 12.93 1.30 - 0.52 14.75 46 3.63 - - 0.54 4.18 47 9.15 5.53 - 0.40 15.08 48 20.16 3.16 - 0.15 23.46 49 - - - 0.25 0.25 50 16.57 1.75 - 0.32 18.64 51 21.56 5.06 - 0.56 27.18 Means 11.01 2.42 2.97 0.56 11.93 SD 6.46 1.35 - 0.68 7.74

-: Under detection limit; *Content of total steroidal < 6.00 mg/g. 2806 Afr. J. Pharm. Pharmacol.

Table 4. Contents of detected compounds in P. polyphylla var. yunnanensis samples (mg/g) from different locations in Southwest China.

Content (mg/g) Polyphyllin I Polyphyllin II Polyphyllin VI Polyphyllin VII Total steroidal saponins Means 9.96 2.68 2.97 0.47 10.88 SD 5.32 1.17 - 0.42 6.62 CV (%) 53.39 43.87 - 89.12 60.86 Lowest 2.90 1.30 - 0.15 0.16 Highest 19.80 4.81 - 2.07 22.07

polyphyllin II and VI were not. Especially, polyphyllin VI polyphyllini> polyphyllin II> polyphyllin VI> polyphyllin VII. has been found only in sample 17 (P. polyphylla var. Polyphyllin I was the main composition of steroidal yunnanensis from Wadu, Baoshan, Yunnan). The mean saponins. The result implied Paris plant may be a new content of total steroidal saponins was 7.74 mg/g, the medicine of anti-cancer drug. There were wide variation highest from P. polyphylla var. pseudothib (Sample 16), of content of bioactive compound in P. polyphylla var. and the lowest from P. cronquistii (Sample 3). The result yunnanensis from different location (Table 4).This might suggested that besides official recorded medicinal Paris have a correlation with the habitat and it need further plants, other species, such as Paris caobangensis, Paris research. mairei, P. polyphylla var. alba, P. polyphylla, and Paris fargesii, would be judged to have medicinal value too. Total steroidal contents of these species were all more ACKNOWLEDGEMENT than 6.00 mg/g (Committee for the Pharmacopoeia of P. R. China, 2010). This work were sponsored by the Special Fund for Agro- Among four compounds, polyphyllin I had the highest scientific Research in the Public Interest (Project no. mean content (11.01 mg/g) followed by polyphyllin VI 201303117). (2.97 mg/g), polyphyllin II, (1.35 mg/g) and polyphyllin VI which had the lowest mean content (0.68 mg/g). It implied that polyphyllin I was the most important part of REFERENCES steroidal saponins in Paris plant. In recent years, strong Calvin CYC (2011). TCM Database@Taiwan: the world's largest anti-cancer effect of polyphyllin I was proved (Ma et al., traditional Chinese medicine database for drug screening in silico. 2009, Man et al., 2010; Kong et al., 2010). Besides the PLoS ONE 6: e15939. traditional efficacy, Paris plants would become a new Committee for the Pharmacopoeia of P.R. China (2010). medicine of anti-cancer drug. P. polyphylla var. Pharmacopoeia of P.R. China, Part I. Guangdong Science and Technology Publishing House and Chemical Industry Publishing yunnanensis, P. polyphylla var. chinensis and other Pairs House, P.R. China. pp. 204-206. genus resources which had high content of polyphyllin I Deng SJ, Yu B, Lou Y (1999). First total synthesis of an exceptional should be paid attention and conserved. potent anti-tumor saponin OSW-1. J. Org. Chem. 64(1):202-208. Most of the bioactive compounds of TCM were Drǎsar P, Moravcove J (2004). Recent advances in analysis of Chinese medical plants and traditional medicines. J. Chromatogr. B. 812:3- secondary metabolites. Quality of TCM depends on the 21. accumulation of these compounds (Su et al., 2005). By Elena C, Marisela T, Jianni X, Sucha S, Dale E J (2010). Interactions comparison (Table 4), our result showed that the ranges between traditional Chinese medicines and Western therapeutics. of contents of bioactive compounds in P. polyphylla var. Curr. Opin. Drug Discov. Devel. 13(1):50-65. Edreva A, Velikova V, Tsonev T, Dagnon S, Gurel A, Aktas l, Gesheva E yunnanensis from different area were wide. Polyphyllin (2008). Stress-protective role of secondary metabolites: diversity of VII had the highest coefficient of variation (CV) which was functions and mechanisms. Gen. Appl. Plant Physiol., 34:67-78. 89.12%, followed by polyphyllin I (53.39) and polyphyllin Huan LQ, Guo LP (2007). Secondary metabolites accumulation and II (43.87). Steroidal saponins varied widely in P. geoherbs formation under environmental stress. China J. Chin Mater. Med. 32(4):277-280. polyphylla var. yunnanensis from different locations. Kong MJ, Fan JQ, Dong AQ, Cheng HF, Xu RZ (2010). Effects of Most bioactive compounds were secondary metabolites polyphyllin I on growth inhibition of human non-small lung cancer (Huang and Guo, 2007). They play an important role in cells and in xenograft. Acta Biochim. Biophys. Sin. 42(11):827-833. adaptation of plant to change of habitat and overcoming Li H (1986). A study on the taxonomy of the genus Paris L. Bull. Bot. Res. 6:109-144. stress constrains (Edreva et al., 2008). The result gave a Li H (1998). The phylogeny of the genus Paris L. In: Li H (ed.), The clue to take further research on the relationship between genus Paris (Trilliaceae). Science Press, Beijing. pp. 8-65. steroidal saponins in Paris and their living environment. Lee MS, Chan YWJ, Kong SK, Yu B, Ooi ECV, Wong NCH, Mak CWT, Fung KP (2005). Effects of polyphyllin D, a steroidal saponin in Paris polyphylla, in growth inhibition of human breast cancer cells and in Conclusion xenograft. Cancer Biol. Ther. 4(11):1248-1254. Ma DD, Lu HX, Xu LS, Xiao W (2009). Polyphyllin D exerts potent anti- tumour effects on Lewis cancer cells under hypoxic conditions. J Int. The content of steroidal saponins in Paris rhizome was Med. Res. 37(3):631-640. Zhang et al. 2807

Man SL, Gao WY, Zhang YJ, Huang LQ, Liu CX (2010). Chemical study Wang GX, Han J, Zhao LW, Jiang DX, Liu YT, Liu XL (2011). and medical application of saponins as anti-cancer agents. Anthelmintic activity of steroidal saponins from Paris polyphylla. Fitoterapia 81(7):703-714. Phytomedicine 17(14):1102-1105. Ong RCY, Lei J, Lee RKY, Cheung JYN, Fung KP, Lin C, Ho HP, Yu B, Zhang JY, Wang YZ, Zhao YL, Yang SB, Zuo ZT, Yang MQ, Zhang J, Li M, Kong SK (2008). Polyphyllin D induces mitochondrial Yang WZ, Yang TM, Jin H (2011). Phytochemicals and bioactivities of fragmentation and acts directly on the mitochondria to induce Paris species. J. Asian Nat. Prod. Res. 13(7):670-681. apoptosis in drug-resistant HepG2 cells. Cancer Lett. 261(2):158- 164. Sun Q, Ju Y, Zhao YF (2002). Steroid saponins with biological activities. Ch. Tradv. Herbal Drugs 33(3):276-279. Su WH, Zhang GF, Li XH, Ou XK (2005). Relationship between accumulation of secondary metabolism in medicinal plant and environmental condition. Chinese Tradit. Herb Drugs 36:1415-1418. Siu FM, Ma DL, Cheung YW, Lok CN, Yan K , Yang ZQ , Yang MS, Xu SX, Ko BCB, He QY, Che CM (2008). Proteomic and transcriptomic study on the action of a cytotoxic saponin (polyphyllin D): induction of endoplasmic reticulum stress and mitochondria-mediated apoptotic pathways. Proteomics 8(15):3105-3117.